目的 探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用.方法 构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2.将H9C2细胞分为阴性对照组(NC)、阳性对照组[MuRF2过表达空载体组(LV-Vector)、MuRF2敲减空载体组(LV-RNAi-Vector)]、MuRF2 过表达组(LV-MuRF2)和 MuRF2 敲减组(LV-RNAi-MuRF2),缺氧培养(5％CO2、1％02、94％N2)后,采用CCK-8法和ELISA法检测心肌细胞损伤水平,利用透射电子显微镜检测心肌细胞超微结构和自噬水平变化,采用Western blot测定自噬相关蛋白LC3-Ⅱ和P62的表达水平.结果 与对照组相比,缺氧组心肌细胞间隙增大、细胞皱缩、多见漂浮的死亡细胞和细胞碎片,心肌细胞活力下降、肌钙蛋白Ⅰ(cTn Ⅰ)含量增加(P均＜0.05);电子显微镜下可观察到部分线粒体发生肿胀,并存在嵴断裂、嵴溶解现象,自噬小体散在胞质内;缺氧组LC3-Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(P均＜0.05).缺氧培养后,与NC组和LV-Vector组相比,LV-MuRF2组细胞活力增加,偶见自噬小体,LC3-Ⅱ蛋白表达水平降低、P62蛋白表达水平增高(P均＜0.05);与NC组和LV-RNAi-Vector组相比,LV-RNAi-MuRF2组细胞活力降低,可见大量自噬小体,LC3-Ⅱ蛋白表达水平升高、P62蛋白表达水平降低(P均＜0.05).结论 泛素连接酶MuRF2可减轻缺氧所致的心肌细胞损伤,其机制可能与抑制心肌细胞线粒体自噬有关.
Ubiquitin Ligase MuRF2 Protects Cardiomyocytes Against Hypoxic Injury by Inhibiting Mitophagy
Objective To define the regulatory effects of ubiquitin ligase muscle ring finger protein 2(MuRF2),a ubiquitin ligase,on the viability and autophagy of hypoxic cardiomyocytes.Methods Rat-original lentivirus vectors of MuRF2 overexpression and knockdown were constructed and infected rat cardiomyocytes H9C2 respectively.The H9C2 cells were divided into negative control(NC)group,positive control(LV-Vector、LV-RNAi-Vector)group,MuRF2 overexpression group(LV-MuRF2),and MuRF2 knockdown group(LV-RNAi-MuRF2).After hypoxic(5％CO2,1％O2,94％N2)culture,cardiomyocyte damages were assessed using the CCK-8 and ELISA assays.Changes in the ultrastructure and autophagy of cardiomyocytes were examined using transmission electron microscopy.The expression levels of autophagy-related proteins LC3-Ⅱ and P62 were de-termined by Western blot.Results Compared with the control group,the hypoxia group exhibited increased in-tercellular gap,cellular shrinkage,and a higher presence of floating dead cells and cell fragments;decreased my-ocardial cell viability,elevated levels of cardiac troponin Ⅰ(cTn Ⅰ)(P all＜0.05).Electron microscopy revealed swollen mitochondria with phenomena such as cristae disruption and dissolution,along with scattered autophagic vacuoles in the cytoplasm.The hypoxia group showed increased expression of LC3-Ⅱ protein and decreased ex-pression of P62 protein(P all＜0.05).After hypoxic culture,compared with the NC group and LV-Vector,cells overexpressing MuRF2 showed increased viability,occasional autophagosomes,de-creased expression levels of LC3-Ⅱ protein,and increased expression levels of P62 protein(P all＜0.05);compared with the NC group and LV-RNAi-MuRF2 group,cells with MuRF2 knockdown exhibited decreased viability,numerous autophagosomes,increased expression levels of LC3-Ⅱ protein,and decreased expression levels of P62 protein(P all＜0.05).Conclusion Ubiquitin ligase MuRF2 alleviates hypoxia-induced cardiomy-ocyte damage,and its role in mitophagy inhibition may be one of the underlying mechanisms.
ubiquitin ligasemuscle ring finger protein 2cardiomyocyteshypoxiamitophagy
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维普
